Cytometers are known in the art that are equipped with up to 7 lasers, which will allow the detection of up to 49 parameters. Independent of these impressive hardware developments, the detection is still based on fluorochromes and is therefore restricted by the need of optical bandpass filters collecting only a small, but characteristic part of a particular emission spectrum. As a consequence, limitations arise in the sensitivity and resolution of partially overlapping fluorescence signals. Moreover, analysis of those high-dimensional datasets, especially in a non-hypothesis driven manner, will be a challenge of future software developments.
Flow cytometry describes a technique where a beam of light (usually laser light) of a single wavelength is directed onto a hydrodynamically-focused stream of fluid. Flow cytometers are frequently used for the analysis of particles such as biological cells or beads in a number of different applications. Such systems allow for determination of both particle morphology and evaluation of particle features by detection of optical labels. The ability to distinguish multiple particles sizes and colors allows multiplex application providing higher capacity of this technology to obtain information from analyzed targets.
The term “particle” as used herein means any discrete target that may be optically analyzed, enumerated or sorted by a flow cytometer. The particles of the present invention include cells, cell fragments and beads. In flow cytometer systems liquid containing target particles are fed from a container into a flow cell. The flow cells separate particles into a stream of individual particles that flow past a detection location. The particles may flow as individual droplets, but to reduce optical noise from refraction it is often preferred to have the particle stream flow through a cuvette where particles in the flow stream are analyzed. At the detection location a beam of focused illumination light (often a laser beam) illuminates the passing particles. Light scattered by the passing particles is detected by forward and side scatter detectors allowing determination of particle morphology. Light emitted from particles is collected and transmitted to detection optics. The particle (generally a cell or bead) may be labelled with one or more dyes having a characteristic excitation and fluorescent emission wavelength. The dye may be conjugated to a binding agent (e.g. a monoclonal antibody) allowing targeting of specific antigen associated with the bead or cell.
Each suspended particle (from especially 0.2 to 150 micrometers) passing through the beam scatters the ray, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source. Light beam splitters separate the collected light into component wavelengths. These beams are directed through a bandpass filter to a light detector (e.g. photomultiplier tube). A specific wavelength associated with each dye is individually detected by at least one detector. The combination of scattered and fluorescent light is picked up by the detectors, and, by analysing fluctuations in brightness at each detector (one for each fluorescent emission peak), it is then possible to derive various types of information about the physical and chemical structure of each individual particle. Forward scatter (FSC) correlates especially with the cell volume and side scatter (SSC) depends on the inner complexity of the particle (i.e., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). Some flow cytometers on the market have eliminated the need for fluorescence and use only light scatter for measurement. Other flow cytometers form images of each cell's fluorescence, scattered light, and transmitted light.
The data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram, or in two-dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed “gates.” Specific gating protocols exist for diagnostic and clinical purposes especially in relation to hematology.
The plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra overlap, signals at the detectors have to be compensated electronically as well as computationally. Data accumulated using the flow cytometer can be analyzed using software, e.g., WinMDI (deprecated), Flowjo, or CellQuest Pro. Once the data is collected, there is no need to stay connected to the flow cytometer. For this reason, analysis is most often done on a separate computer. This is especially necessary in core facilities where usage of these machines is in high demand.
Recent progress on automated population identification using computational methods has offered an alternative to traditional gating strategies. Automated identification systems could potentially help findings of rare and hidden populations. Representative automated methods include FLOCK in Immunology Database and Analysis Portal (ImmPort), FLAME in GenePattern and flowClust in Bioconductor. Collaborative efforts have resulted in an open project called FlowCAP (Flow Cytometry: Critical Assessment of Population Identification Methods) to provide an objective way to compare and evaluate the flow cytometry data clustering methods, and also to establish guidance about appropriate use and application of these methods.
In a conventional flow cytometer a combination of optical filters and dichroics is used in order to detect a specific fluorescence emission range of a single dye. In a multicolour experiment the spill over needs to be compensated and it has to be carefully analysed by sequential gating strategies. Therefore, actually multi-dimensional experiments, are still a time-consuming procedure including cytometer setup, sample collection and manual data analysis. Moreover, multidimensionality is restricted by the availability of appropriate fluorochromes that can be combined according to their unique emission spectra characteristics.